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1.  Reducing the Frequency of Acute Otitis Media by Individualized Care 
Objective
We sought to determine if use of more stringent diagnostic criteria for acute otitis media (AOM) than currently advocated by the American Academy of Pediatrics (AAP), tympanocentesis and pathogen-specific antibiotic treatment (individualized care) would result in reducing the incidence of recurrent AOM and consequent tympanostomy tube surgery.
Methods
A 5 year longitudinal, prospective study in Rochester NY was conducted from July 2006 – July 2011 involving 254 individualized care children. When this individualized care group developed symptoms of AOM, strict diagnostic criteria were applied and a tympanocentesis was performed. Pathogen resistance to empiric high dose amoxicillin/clavulanate (80mg/kg of amoxicillin component) caused a change in antibiotic to an optimized choice. Legacy controls (n=208) were diagnosed with the same diagnostic criteria by the same physicians as the individualized care group and received the same empiric amoxicillin/clavulanate (80mg/kg of Amoxicillin component) but no tympanocentesis or change in antibiotic. Community control children (n=1020) were diagnosed according to current AAP guidelines and treated with high dose amoxicillin (80 mg/kg) without tympanocentesis as guideline recommended.
Results
5.9% of children of the individualized care group compared to 14.4% of Legacy controls and 27.3% of community controls became otitis prone (OP), defined as 3 episodes of AOM within a 6-month time span or 4 AOM episodes within a 12-month time span (p<0.0001). 2.4% of the individualized care group compared to 6.3% of Legacy controls, and 14.8% of community controls received tympanostomy tubes (p<0.0001).
Conclusions
Individualized care of AOM significantly reduces the frequency of AOM and tympanostomy tube surgery. Use of strict diagnostic criteria for AOM and empiric antibiotic treatment using evidence-based knowledge of circulating otopathogens and their antimicrobial susceptibility profile also produces improved outcomes.
doi:10.1097/INF.0b013e3182862b57
PMCID: PMC3681924  PMID: 23340550
otitis prone; acute otitis media; tympanostomy tubes; tympanocentesis; amoxicillin; amoxicillin/clavulanate
2.  CD4+ T-cell Responses Among Adults and Young Children In Response to Streptococcus pneumoniae and Haemophilus influenzae Vaccine Candidate Protein Antigens 
Vaccine  2013;31(30):3090-3097.
Summary
We characterized cytokine profiles of CD4+ T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and Protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 - and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4+ T cells producing IL-2, (p=0.004). Vaccine antigen-specific CD4+ T-cell populations in adults were largely of effector (TEM) and/or central memory (TCM) phenotypes as defined by CD45RA−CCR7+ or CD45RA−CCR7− respectively; however among young children antigen-specific IL-2 producing CD4+ T cells demonstrated CD45RA+ expression (non-memory cells). We conclude that adults have circulating memory CD4+T cells (CD45RA−) that can be stimulated by all the tested Spn and NTHi protein vaccine candidate antigens, whereas young children have a more limited response.
doi:10.1016/j.vaccine.2013.03.060
PMCID: PMC3777711  PMID: 23632305
Streptococcus pneumonia; non-typeable Haemophilus influenzae; CD4+ T-helper (h) cell; IFN-y; IL-2; memory T cells
3.  Higher serum levels of interleukin 10 occur at onset of acute otitis media caused by Streptococcus pneumoniae compared to Haemophilus influenzae and Moraxella catarrhalis 
The Laryngoscope  2013;123(6):1500-1505.
Objective
Acute otitis media (AOM) involves an inflammatory response to microbes in the middle ear that facilitates clearance of otopathogens. Clinically, Streptococcus pneumoniae (Spn) infections of the respiratory tract are characterized by greater inflammatory responses than nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat). Interleukin 10 (IL-10) plays an important role in down-regulating the inflammatory response. We compared serum IL-10 levels in children before onset, at onset and after recovery from AOM caused by Spn, NTHi, and Mcat. We sought to determine if IL-10 could serve as a biomarker to distinguish AOM caused by Spn versus NTHi and Mcat.
Study Design
Prospective, longitudinal study in a primary care pediatric practice in Rochester, NY.
Methods
Participants were 54 children 6 to 30 months of age. Outcomes measured were serum IL-10 levels when healthy, at onset of AOM and after recovery from AOM.
Results
Serum IL-10 was elevated when children developed AOM (p=0.013) due to infections caused by Spn (p= 0.011) but not AOM caused by NTHi or Mcat. Middle ear fluid levels of IL-10 mirrored those seen in serum but were 10-fold higher (p=0.02). Other effector cytokines in serum: IL-4, IFN-γ and TNF-alpha, did not show the same increases as IL-10 at onset of AOM.
Conclusion
Our study indicates that AOM caused by Spn elicits a significantly higher IL-10 response compared to NTHi and Mcat and may prove to be a biomarker of AOM infections by Spn.
doi:10.1002/lary.23973
PMCID: PMC3966296  PMID: 23404508
Interleukin 10; acute otitis media; Streptococcus pneumoniae; Haemophilus influenzae; Moraxella catarrhalis; cytokines
4.  Comparison of Amoxicillin/Clavulanate High Dose to Cefdinir in Treatment of Acute Otitis Media 
Drugs  2012;72(15):1991-1997.
Objective
To compare the clinical efficacy of amoxicillin/clavulanate high dose (Amox/clav HD) as 10 days therapy to cefdinir as 5 days therapy for acute otitis media (AOM).
Study Design
Diagnosis of AOM was based on specific criteria by validated otoscopists at 2 AOM research centers. The outcome measure was resolution of all symptoms and signs of AOM except persistence of middle ear effusion at test-of-cure (TOC) 12-15 days after antibiotic treatment .
Results
330 children (x = 13.1 months) with AOM were studied. At TOC 256 children were cured, 69 failed and 5 were lost to follow-up. Amox/clav HD-treated children had a better cure rate (86.5%) than cefdinir (71.0%), p=0.001. Clinical outcomes showed that amox/clav HD was correlated with more frequent cure outcomes and that cefdinir was correlated with less frequent cure outcomes as children increased in age between 6 and 24 months.
Conclusion
When children have bona fide AOM an assessment of outcome is judged by validated otoscopists. 10 days amox/Clav HD is significantly more effective than 5 days of cefdinir as therapy for AOM. A trial comparing 10 days of cefdinir may have led to a different conclusion.
doi:10.2165/11590320-000000000-00000
PMCID: PMC3963277  PMID: 23039319
Acute otitis media; Scoring systems; amoxicillin; cefdinir; antibiotics; clinical trial
5.  Transcriptome signature in young children with acute otitis media due to non-typeable Haemophilus influenzae  
International Immunology  2013;25(6):353-361.
The transcriptome during acute otitis media due to NTHi shows relatively weak immune activation
Non-typeable Haemophilus influenzae (NTHi) causes acute otitis media (AOM) in young children. In our recent paper in Microbes and Infection we described the transcriptome signature elicited from PBMCs at onset of AOM caused by Streptococcus pneumoniae. In the current study we found very different results with NTHi AOM infections; 5.1% of 29 187 genes were differentially regulated by more than 2-fold at the onset of AOM compared with the pre-infection healthy state in the same children. Among the 1487 transcripts, 100 genes associated with the immune defense response were specifically analyzed. About half of the differentially regulated genes associated with antibacterial activity and the cell-mediated immune response were activated and half were suppressed. The important signatures for NTHi in children suggested that the balance of the immune response was toward suppression. Moreover, 90% of the genes associated with a pro-inflammatory cytokine response were down-regulated. The genes associated with the classic complement pathway were down-regulated, although the alternative complement pathway genes were up-regulated. These results provide the first human transcriptome data identifying gene expression in the immune response to be predominantly down-regulated at the onset of AOM due to NTHi.
doi:10.1093/intimm/dxs154
PMCID: PMC3708516  PMID: 23411728
acute otitis media; immune response signatures; non-typeable Haemophilus influenzae; transcriptome profile
6.  Antibody Response to Haemophilus influenzae Outer Membrane Protein D, P6, and OMP26 After Nasopharyngeal Colonization and Acute Otitis Media in Children 
Vaccine  2010;28(44):7184-7192.
Development of natural antibodies to 3 nontypeable Haemophilus influenzae (NTHi) outer membrane proteins (D, P6 and OMP26) was prospectively studied in 168 children 6–30 months of age during NP colonization and AOM. IgG antibody to protein D, P6 and OMP26 increased with age (p<0.001). Serum IgG responses to NP colonization were different for the 3 proteins: protein D responses occurred at a later age than P6, and OMP26 responses were minimal. For all 3 proteins serum antibody levels in the convalescent phase of AOM infection were not as high as after NP colonization. Antibodies to protein D and P6 but not OMP26 were bactericidal.
doi:10.1016/j.vaccine.2010.08.063
PMCID: PMC3959881  PMID: 20800701
Non-typeable Haemophilus influenzae; P6 protein; protein D; protein OMP26; Acute otitis media; carriage
7.  New Patterns in the Otopathogens Causing Acute Otitis Media Six to Eight Years After Introduction of Pneumococcal Conjugate Vaccine 
Objective
To describe NP and AOM otopathogens during the time frame 2007-2009, six to eight years after the introduction of 7-valent pneumococcal conjugate (PCV7) in the US and to compare nasopharyngeal (NP) colonization and acute otitis media (AOM) microbiology in children 6 to 36 months of age having 1st and 2nd AOM episodes with children who are otitis prone.
Methods
Prospectively, the microbiology of NP colonization and AOM episodes was determined in 120 children with absent or infrequent AOM episodes. NP samples were collected at 7 routine visits between 6 and 30 months of age and at the time of AOM. For 1st and subsequent AOM episodes, middle ear fluid (MEF) was obtained by tympanocentesis. Eighty otitis prone children were comparatively studied. All 200 children received age-appropriate doses of PCV7.
Results
We found PCV7 serotypes were virtually absent: (0.9% isolated from both NP and MEF) in both study groups. However, non-PCV7 serotypes replaced PCV serotypes such that the frequency of isolation of S. pneumoniae (Spn) was nearly equal to that of non-typeable Haemophilus influenzae (NTHi). M. catarrhalis (Mcat) was less common and Staphylococcus aureus infrequent in the NP and MEF from the two groups. The proportion of Spn, NTHi and Mcat causing AOM was similar in children with 1st and 2nd AOM episodes compared to otitis prone children. However, oxacillin-resistant Spn isolated from the NP and MEF was 19% for the absent/infrequent and 58% for the otitis prone groups, p<0.0001. Beta-lactamase producing NTHi occurred more frequently in the otitis prone group, p=0.04.
Conclusions
Six to 8 years after widespread use of PCV7, Spn strains expressing vaccine-type serotypes have virtually disappeared from the NP and MEF of vaccinated children. NP colonization and AOM has changed to non-PCV7 strains of Spn. NTHi continues to be a major AOM pathogen. The otopathogens in 1st and 2nd AOM and in otitis prone children are very similar although Spn and NTHi are more often antibiotic resistant in the otitis prone.
doi:10.1097/INF.0b013e3181c1bc48
PMCID: PMC3959886  PMID: 19935445
Nasopharyngeal; AOM; S. pneumoniae; H. influenzae; M. catarrhalis
8.  PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans 
Streptococcus pneumoniae (pneumococci) adhere to human nasopharyngeal (NP) epithelial cells as a first step in pathogenesis and adherence of pneumococci to lung epithelia may be required to establish pneumonia. We sought to determine if PcpA can serve as an adhesin to human NP (D562) and lung (A549) epithelial cells and whether PcpA mediated adherence can be inhibited by human anti-PcpA antibodies. A PcpA isogenic mutant was prepared on a wild type pneumococcal TIGR4 background. When the mutant and wild type strains were compared for adherence to D562 and A549 cell lines a reduction in adherence by the mutant was observed (p= 0.0001 for both cell types). PcpA was ectopically expressed on the surface of minimally-adherent heterologous host E coli resulting in augmented adherence to D562 (p= 0.002) and A549 (p= 0.015) cells. Total IgG was purified from a pool of 6 human sera having high IgG titers of anti-pneumococcal proteins. The purified IgG reduced TIGR4 adherence to D562 cells but we determined that this effect was largely due to bacterial cell aggregation as determined by flow cytometry and confocal microscopy. Fab fragments were prepared from pooled IgG sera. Inhibition of TIGR4 adherence to D562 cells was observed using the Fab fragments without causing bacterial aggregation (p=0.0001). Depletion of PcpA-specific Fab fragments resulted in an increase in adherence of TIGR4 to D562 cells (p=0.028). We conclude that PcpA can mediate adherence of pneumococci to human NP and lung epithelial cells and PcpA mediated adherence can be inhibited by human anti-PcpA antibodies.
doi:10.1016/j.micinf.2012.06.007
PMCID: PMC3490615  PMID: 22796387
9.  Nontypeable Streptococcus pneumoniae as an Otopathogen 
Among 34 Spn sequential isolates from middle ear fluid we found a case of a nontypeable Streptococcus pneumoniae (NT-Spn) in a child with AOM. The strain was pneumolysin PCR positive and capsule gene PCR negative. Virulence of the NT-Spn was confirmed in a chinchilla model of AOM.
doi:10.1016/j.diagmicrobio.2010.09.019
PMCID: PMC3897191  PMID: 21251566
multi-locus sequence typing; nontypeable Streptococcus pneumoniae; acute otitis media
10.  Dual Orientation of the Outer Membrane Lipoprotein P6 of Nontypeable Haemophilus influenzae 
Journal of Bacteriology  2013;195(14):3252-3259.
The majority of outer membrane (OM) lipoproteins in Gram-negative bacteria are tethered to the membrane via an attached lipid moiety and oriented facing in toward the periplasmic space; a few lipoproteins have been shown to be surface exposed. The outer membrane lipoprotein P6 from the Gram-negative pathogenic bacterium nontypeable Haemophilus influenzae (NTHi) is surface exposed and a leading vaccine candidate for prevention of NTHi infections. However, we recently found that P6 is not a transmembrane protein as previously thought (L. V. Michel, B. Kalmeta, M. McCreary, J. Snyder, P. Craig, M. E. Pichichero, Vaccine 29:1624–1627, 2011). Here we pursued studies to show that P6 has a dual orientation, existing infrequently as surface exposed and predominantly as internally oriented toward the periplasmic space. Flow cytometry using three monoclonal antibodies with specificity for P6 showed surface staining of whole NTHi cells. Confocal microscopy imaging confirmed that antibodies targeted surface-exposed P6 of intact NTHi cells and not internal P6 in membrane-compromised or dead cells. Western blots of two wild-type NTHi strains and a mutant NTHi strain that does not express P6 showed that P6 antibodies do not detect a promiscuous epitope on NTHi. Depletion of targets to nonlipidated P6 significantly decreased bactericidal activity of human serum. Protease digestion of surface-exposed P6 demonstrated that P6 is predominantly internally localized in a manner similar to its homologue Pal in Escherichia coli. We conclude that P6 of NTHi is likely inserted into the OM in two distinct orientations, with the predominant orientation facing in toward the periplasm.
doi:10.1128/JB.00185-13
PMCID: PMC3697637  PMID: 23687267
11.  Vaccine Candidates PhtD and PhtE of Streptococcus pneumoniae are adhesins that elicit functional antibodies in humans 
Vaccine  2012;30(18):2900-2907.
We evaluated the role of vaccine candidate surface proteins, PhtD and PhtE as antigens with functional importance for Streptococcus pneumoniae (pneumococci) in adherence to nasopharyngeal (D562) and lung (A549) epithelial cell lines. Comparing TIGR4 to PhtD and PhtE-deleted isogenic mutants, a 40% (p=0.001) and 42% (p=0.002) drop in the number of epithelial cells with adherent pneumococci was observed to both cells lines with the mutants, as quantitated using flow cytometry. We expressed PhtD and PhtE on the surface of E coli and demonstrated that when PhtD and PhtE were surface expressed on E coli adherence increased to D562 and A549 cells, compared with the E coli parent strain (p = 0.005, 0.013 for D562 and p=0.034, p=0.035 for A549). Using flow cytometry and confocal microscopy we found that pneumococci aggregated in the presence of human serum IgG, leading to a non-specific drop in adherence. Therefore IgG Fab fragments were prepared to study the functional role of PhtD and PhtE-specific Fabs in blocking adherence. The addition of 1 μg of IgG Fab from adult sera led to a 34% reduction (p= 0.002) and from children a 20% (p= 0.023) reduction in D562 epithelial cells with adherent pneumococci. In purified IgG from adult sera, the depletion of PhtD and PhtE specific Fab from total IgG Fab resulted in a significant increase in the number of D562 epithelial cells with adherent pneumococci (p=0.005 for PhtD and p=0.024 for PhtE). We conclude that antibody directed to PhtD and PhtE are adhesins of pneumococci, if raised by vaccination, may function to prevent pneumococcal adherence to human airway epithelial cells.
doi:10.1016/j.vaccine.2012.02.023
PMCID: PMC3490617  PMID: 22349524
12.  Reduced Serum IgG Responses to Pneumococcal Antigens in Otitis-Prone Children May Be Due to Poor Memory B-Cell Generation 
The Journal of Infectious Diseases  2012;205(8):1225-1229.
A low level of serum antibody to antigens expressed by Streptococcus pneumoniae has been proposed to explain the susceptibility of children to recurrent episodes of acute otitis media (hereafter, “otitis-prone children”). By use of enzyme-linked immunospot assays, the percentages of memory B cells to pneumococcal protein antigens PhtD, LytB, PcpA, PhtE, and Ply were compared between otitis-prone and non–otitis-prone children at the time of acute otitis media or nasopharyngeal colonization with S. pneumoniae. We found significantly lower percentages of memory B cells to 3 pneumococcal protein antigens (PhtD, PhtE, and Ply) and reduced antigen-specific immunoglobulin G concentrations in otitis-prone children, compared with non–otitis-prone children.
doi:10.1093/infdis/jis179
PMCID: PMC3308907  PMID: 22383675
13.  Clinical Significance of Serum S100A12 in Acute Otitis Media in Young Children 
Background
S100A12 is a calcium-binding protein predominantly expressed in neutrophil granulocytes in response to infections or inflammation. Acute otitis media (AOM) is a local infection mainly caused by Streptococcus pneumoniae (Spn), nontypeable Haemophilus influenzae (NTHi) and/or Moraxella catarrhalis (Mcat).
Methods
To study if S100A12 values could serve as a diagnostic marker, serum S100A12 concentrations were tested in young children before, at onset and after recovery from AOM.
Results
Among 116 children with AOM we found that serum S100A12 concentrations were significantly increased at onset of AOM compared to before infection (p=0.0001), and returned to normal levels when the children recovered from the infection (p=0.01). Elevation of S100A12 correlated with the presence of Spn (p=0.004) or NTHi (p=0.04) in the middle ear, but not with Mcat or upper respiratory viral infection. Change in serum value of S100A12 at onset of AOM were not related to the frequency of occurrence of AOM or the age of the child.
Conclusion
S100A12 may be a useful biomarker for onset of AOM due to Spn and NTHi; and for following children with AOM.
doi:10.1097/INF.0b013e31824672cb
PMCID: PMC3308144  PMID: 22198825
S100A12; acute otitis media; biomarker; young children
14.  Phylogenetic relatedness and diversity of non-typable Haemophilus influenzae in the nasopharynx and middle ear fluid of children with acute otitis media 
Journal of Medical Microbiology  2011;60(Pt 12):1841-1848.
The phylogenetic relationships of non-typable Haemophilus influenzae (NTHi) strains prospectively isolated from healthy children and children with acute otitis media (AOM) were analysed using multilocus sequence typing (MLST). A total of 165 NTHi isolates were collected over a 3.5 year time frame during 2006 through 2009. The strains were tested for β-lactamase production; 28.5 % were positive. Seventy different NTHi sequence types (STs) were identified of which 29 (41.4 %) were novel. NTHi strains did not show any phylogenetic grouping or clustering among asymptomatic colonizing strains or strains that caused AOM, or based on β-lactamase enzyme production. Evaluation of triplets and other siblings over time demonstrated relatively frequent genetic exchanges in NTHi isolates in vivo in a short time frame and subsequent transfer among children in a family. Comparison of the MLST STs isolated at different time points showed that in ~85 % of the nasopharynx (NP) colonizations, NTHi strains cleared from the host within 3 months, that sequential colonization in the same child involved different strains in all cases except one, and that NP and middle ear isolates were identical STs in 84 % of cases. In this first study of its type to our knowledge, we could not identify predominant MLST types among strains colonizing the NP versus those causing AOM or expressing a β-lactamase enzyme conferring penicillin resistance in children.
doi:10.1099/jmm.0.034041-0
PMCID: PMC3347915  PMID: 21799196
15.  Reduced Memory CD4+ T-Cell Generation in the Circulation of Young Children May Contribute to the Otitis-Prone Condition 
The Journal of Infectious Diseases  2011;204(4):645-653.
Background. An explanation for the immunologic dysfunction that causes children to be prone to repeated episodes of acute otitis media (AOM) has long been sought. Poor antibody response has been associated with the otitis-prone condition; however, there is no precise mechanistic explanation for this condition.
Methods. Non–otitis-prone and otitis-prone children with AOM or nasopharyngeal (NP) colonization caused by either Streptococcus pneumoniae or Haemophilus influenzae were compared for pathogen-specific CD4+ T-helper memory responses by stimulating peripheral blood mononuclear cells using 6 vaccine candidate S. pneumoniae and 3 H. influenzae protein antigens. Samples were analyzed by multi-parameter flow cytometry.
Results. Significantly reduced percentages of functional CD45RALow memory CD4+ T cells producing specific cytokines (interferon γ, interleukin [IL]–2, IL-4 and IL-17a) were observed in otitis-prone children following AOM and NP colonization with either S. pneumoniae or H. influenzae. Immunoglobulin (Ig) G responses to the studied protein antigens were reduced, which suggests that antigen-specific B-cell function may be compromised as a result of poor T-cell help. Staphylococcal enterotoxin B stimulated similar cytokine patterns in memory CD4+T cells in both groups of children.
Conclusions. Otitis-prone children have suboptimal circulating functional T-helper memory and reduced IgG responses to S. pneumoniae or H. influenzae after colonization and after AOM; this immune dysfunction causes susceptibility to recurrent AOM infections.
doi:10.1093/infdis/jir340
PMCID: PMC3144174  PMID: 21791667
16.  Serum Antibody Response to Five Streptococcus pneumoniae Proteins during Acute Otitis Media in Otitis Prone and Non-Otitis Prone Children 
Background
Streptococcus pneumoniae (Spn) is one of the common bacteria responsible for episodic acute otitis media (AOM; non-otitis prone), recurrent AOM (otitis-prone) and AOM treatment failure (AOMTF) in children.
Objective
From a population of 268 children we sought to compare the serum IgG antibody titers to five different Spn proteins (PhtD, LytB, PcpA, PhtE and Ply) that are vaccine candidates in children with episodic AOM (n=34), who were otitis prone (n=35), and who had AOMTF (n=25) caused by Spn.
Methods
Antibody was quantitated by ELISA.
Results
At their acute AOM visit, anti-PhtD, -LytB, -PhtE and −Ply IgG antibody titers in otitis-prone children were significantly lower compared to non-otitis prone children (p <0.05) and children with AOMTF (p <0.05). Comparing acute to convalescent titers of antibody after AOM we found that otitis-prone, AOMTF and non-otitis prone children had no significant change in geometric mean IgG antibody titers against the five proteins (except for PhtE in children with AOMTF), but detailed analysis showed that about one-third of the children in each cohort had a 2-fold rise in antibody to the studied antigens. While non-otitis prone children had significant increases (p <0.001) between 6 and 24 months of age in anti-PhtD, PcpA, PhtE and Ply IgG antibody titers as a consequence of nasopharyngeal colonization and AOM, otitis-prone children either failed to show rises or the rises were significantly less than the non-otitis prone children.
Conclusion
Otitis-prone and AOMTF children mount less of an IgG serum antibody response than non-otitis prone children to Spn proteins following AOM and nasopharyngeal colonization.
doi:10.1097/INF.0b013e31821c2d8b
PMCID: PMC3296524  PMID: 21487325
S. pneumoniae; PhtD; PhtE; Ply; PcpA; LytB; Acute otitis media; Otitis-prone
17.  Vaccine Candidate P6 of Nontypable Haemophilus influenzae is Not a Transmembrane Protein Based on Protein Structural Analysis 
Vaccine  2011;29(8):1624-1627.
P6 has been a vaccine candidate for nontypable Haemophilus influenzae (NTHi) based on its location on the outer membrane and immunogenicity. Because P6 is attached to the inner peptidoglycan layer of NTHi, and is putatively surface exposed, it must be a transmembrane protein. We examined the P6 structure using computational modeling, a P6 modified by site-directed mutagenesis to study monoclonal antibody attachment to P6, and nuclear magnetic resonance spectroscopy. We found that P6 cannot be a transmembrane protein, and therefore may not be surface exposed. We conclude that there may be another protein on the surface of NTHi that has epitopes similar if not identical to P6.
doi:10.1016/j.vaccine.2010.12.082
PMCID: PMC3070289  PMID: 21215345
nontypable Haemophilus influenzae; P6 protein; outer membrane protein
18.  Serum Antibody Response to Three Non-typeable Haemophilus influenzae Outer Membrane Proteins During Acute Otitis Media and Nasopharyngeal Colonization in Otitis Prone and Non-Otitis Prone Children 
Vaccine  2010;29(5):1023-1028.
Non-typeable Haemophilus influenzae (NTHi) is the most common bacteria responsible for episodic acute otitis media (AOM; non-otitis prone), recurrent AOM (rAOM; otitis prone) and AOM treatment failure (AOMTF) in children. In this 3.5 years of prospective study, we measured the serum antibody response to outer membrane proteins D, P6 and OMP26 of NTHi in children with AOM (n= 26), rAOM (n= 32), AOMTF (n=27). The geometric mean titers (GMTs) of IgG at their acute AOM visit against protein D in otitis prone children were significantly lower compared to AOMTF (p value < 0.01) and non-otitis prone (p value <0.03) children; otitis prone children had significantly lower IgG levels to P6 compared to AOMTF children (p value < 0.02); otitis prone children had significantly lower IgG levels to OMP26 compared to AOMTF children (p value <0.04). Comparing acute to convalescent titers after AOM, otitis prone and AOMTF children had no significant change in total IgG against all the three proteins, while non-otitis prone children had significant increases to protein D. Anti-Protein D, P6 and OMP26 antibody levels measured longitudinally during NP colonization between age 6 and 24 months in 10 otitis prone children and 150 non-otitis prone children showed < 2-fold increases over time in otitis prone children compared to > 4 fold increases in the non-otitis prone children (p value < 0.001). We conclude that otitis prone children mount less of an IgG serum antibody response toward Protein D, P6 and OMP26 after AOM which may account for recurrent infections. The data on acute sera of otitis prone versus non-otitis prone children and the acute-to-convalescence response in non-otitis prone children point to a possible link of anti-PD to protection. Moreover, the data suggest that otitis prone children should be evaluated for their responses to Protein D, P6 and OMP26 vaccine antigens of NTHi.
doi:10.1016/j.vaccine.2010.11.055
PMCID: PMC3042269  PMID: 21129398
Non-typeable Haemophilus influenzae; P6 protein; protein D; protein OMP26; Acute otitis media; Otitis prone
19.  Antibody response to Streptococcus pneumoniae proteins PhtD, LytB, PcpA, PhtE and Ply after nasopharyngeal colonization and acute otitis media in children 
We prospectively compared serum antibody levels of 5 Streptococcus pneumoniae (Spn) proteins: PcpA PhtD, PhtE Ply and LytB associated with nasopharyngeal (NP) colonization and acute otitis media (AOM) infection in a cohort of 6–30 mo old children. Antigen-specific antibody titers were determined by ELISA. A total of 731 visits among 168 children were studied. There were 301 Spn NP colonization episodes documented in 109 (65%) children and 42 Spn AOM episodes in 34 (20%) children. IgG antibody titers to the 5 proteins were significantly different among children over time (p < 0.001), with a rank order as follows: PcpA > PhtE = PhtD > Ply > LytB Characterization of IgG and IgM acute and convalescent serum antibody levels of Spn AOM infection showed the kinetics of the response differed among children, with the same rank order of antibody levels over time. Individual data showed that some children responded to AOM with an antibody increase to one or more of these Spn proteins but some children failed to respond. We conclude that antibody levels to Spn proteins PcpA PhtD, PhtE, Ply and LytB, all rise over time in children age 6 to 30 mo following natural exposure to Spn after NP colonization and AOM; however, there were significant differences in quantity of antibody elicited among these potential vaccine antigens.
doi:10.4161/hv.19820
PMCID: PMC3679215  PMID: 22495112
Acute otitis media; LytB; PcpA; PhtD; PhtE; Ply; Streptococcus pneumoniae
20.  Antibody in Middle Ear Fluid of Children Originates Predominantly from Sera and Nasopharyngeal Secretions 
Clinical and Vaccine Immunology : CVI  2012;19(10):1593-1596.
The human middle ear is devoid of any immunocompetent cells in normal mucosa. We sought to determine the source of antibody present in the middle ear of children. Total IgG, IgA, and secretory IgA antibodies were determined by enzyme-linked immunosorbent assay from the nasopharyngeal, middle ear, and serum samples of children with acute otitis media. The two-dimensional gel electrophoresis pattern of the entire array of IgA antibodies in the nasal wash (NW) and middle ear fluid (MEF) was compared from the MEF and NW samples using isoelectric focusing and Western blotting. The total IgG and IgA antibodies in the MEF and NW samples of 137 children were compared. The ratio of IgG to IgA in the MEF was significantly different (P < 0.008) compared to NW because IgA levels were higher and IgG levels lower in NW. The IgG/IgA ratio of MEF resembled serum consistent with transudation to the MEF. Small amounts of secretory IgA were detected in MEF but the electrophoresis patterns of the entire array of IgA antibodies in the MEF and NW were virtually identical in each child evaluated; thus, IgA in MEF derived predominantly from serum and the nasopharynx by reflux via the Eustachian tube. The IgG/IgA antibody levels in the MEF and the same composition of IgA antibody in the MEF and NW identifies the predominant source of antibody in the MEF as a transudate of serum combined with nasal secretions refluxed from the nasopharynx in children.
doi:10.1128/CVI.05443-11
PMCID: PMC3485881  PMID: 22855395
21.  Simultaneous Assay for Four Bacterial Species Including Alloiococcus otitidis Using Multiplex-PCR in Children With Culture Negative Acute Otitis Media 
Background
The 3 most commonly encountered bacteria in acute otitis media (AOM) are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Conventional culture methods detect these pathogens in only 60% to 70% of cases of AOM. Alloiococcus otitidis, another potential pathogen, has often been ignored.
Methods
Tympanocentesis was performed in 97 children with AOM presenting with a bulging tympanic membrane (TM) producing 170 middle ear fluids (MEFs). S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis were isolated in 21%, 32%, 8%, and 0% of MEFs, respectively; no otopathogen was isolated in 29% of MEFs. In nasopharyngeal cultures at the time of AOM diagnosis, 34%, 36%, 17%, and 0% and in oropharyngeal cultures, 7%, 31%, 11%, and 0% grew S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis, respectively. No otopathogen was isolated in 23% of nasopharyngeal and 20% of oropharyngeal cultures. Multiplex polymerase chain reaction (PCR) was used to detect DNA of the 4 bacterial species in culture negative samples.
Results
All culture-positive MEF, nasopharyngeal and oropharyngeal samples tested were also multiplex-PCR positive, indicating the reliability of the method. Culture-negative samples of MEF from children with a bulging TM yielded S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis DNA in 51%, 35%, 14%, and 32% of MEF, in 45%, 31%, 10%, and 9% of nasopharyngeal and in 31%, 23%, 0%, and 3% of oropharyngeal, respectively. In 9% of the cases A. otitidis DNA was found without detection of a second organism in MEF.
Conclusions
Conventional culture detected otopathogens in MEF of children with a bulging TM in 71%; using multiplex-PCR, otopathogens were detected in 88% of MEF (P < 0.01). Similar improved detection of otopathogens was noted with nasopharyngeal and oropharyngeal cultures.
doi:10.1097/INF.0b013e3181d9e639
PMCID: PMC3581301  PMID: 20335823
acute otitis media; Alloiococcus otitidis; multiplex-PCR; otopathogen; Streptococcus pneumoniae
22.  Identification of Streptococcus pneumoniae and Haemophilus influenzae in culture-negative middle ear fluids from children with acute otitis media by combination of multiplex PCR and multi-locus sequencing typing 
Objective
Streptococcus pneumoniae (Spn) and Haemophilus influenzae (Hflu) are major etiologic pathogens for acute otitis media (AOM). However, when Spn and Hflu strains are not identified by traditional culture methods, use of alternative PCR-based diagnosis becomes critical. This study aimed to develop a combined molecular method to accurately detect these otopathegens.
Methods
Middle ear fluid (MEF) samples were collected by tympanocentesis from children with AOM to isolate Spn and Hflu by standard culture procedures. Multiplex PCR (mPCR) and multi-locus sequence typing (MLST) techniques were used to detect Spn and Hflu in culture-negative MEF samples.
Results
We found 20 Spn or Hflu culture-positive MEF samples that were mPCR-positive and typeable by MLST. The sequences of the housekeeping genes and the MLST allelic profiles obtained from Spn or Hflu culture isolates matched exactly MEF samples that were tested directly without culture isolation. Of 63 MEF samples that were culture-negative for Spn, 38% (24/63) were mPCR-positive for Spn. Of 50 MEF samples that were culture-negative for Hflu, 24% (12/50) were mPCR-positive for Hflu. Among these culture-negative but mPCR-positive MEF samples, 25% (6/24) and 25% (3/12) were typeable by MLST for Spn and Hflu, respectively.
Conclusions
MEF samples may be analyzed with mPCR and MLST directly without culture isolation and the addition of mPCR and MLST may accurately identify Spn and Hflu in MEF of children with AOM when bacterial culture is negative.
doi:10.1016/j.ijporl.2010.11.008
PMCID: PMC3563323  PMID: 21126776
23.  Nasopharyngeal Bacterial Interactions in Children 
Emerging Infectious Diseases  2012;18(11):1738-1745.
TOC summary: Pathogen prevalence differs during periods of health and at onset of acute otitis media.
doi:10.3201/eid1811.111904
PMCID: PMC3559157  PMID: 23092680
Streptococcus pneumoniae; Haemophilus influenzae; Moraxella catarrhalis; Staphylococcus aureus; bacteria; nasopharyngeal bacterial infections; polymicrobial colonization; bacterial interaction; acute otitis media; children
24.  Breastfeeding is associated with a reduced frequency of acute otitis media and high serum antibody levels against NTHi and outer membrane protein vaccine antigen candidate P6 
Pediatric research  2009;66(5):565-570.
Nontypeable Haemophilus influenzae (NTHi) causes acute otitis media (AOM) in infants. Breast-feeding protects against AOM and/or nasopharyngeal (NP) colonization; however, the mechanism of protection is incompletely understood. Children with AOM and healthy children were studied according to feeding status: breast-fed, breast/formula fed or formula fed. Cumulative episodes of AOM, ELISA titers of serum IgG antibodies to whole-cell NTHi and vaccine candidate outer membrane protein P6, bactericidal titers of serum and NP colonization by NTHi were assessed. A lower incidence of AOM was found in breast versus formula fed children. Levels of specific serum IgG antibody to NTHi and P6 were highest in breast fed, intermediate in breast/formula fed and lowest in formula fed infants. Serum IgG antibody to P6 correlated with bactericidal activity against NTHi. Among children with AOM, the prevalence of NTHi in the NP was lower in breast versus non-breast fed infants. We conclude that breast-feeding shows an association with higher levels of antibodies to NTHi and P6, suggesting that breast-feeding modulates the serum immune response to NTHi and P6. Higher serum IgG might facilitate protection against AOM and NP colonization in breast-fed children.
doi:10.1203/PDR.0b013e3181b4f8a6
PMCID: PMC2783794  PMID: 19581824
25.  Mercury Levels in Premature and Low Birth Weight Newborns after Receipt of Thimerosal-Containing Vaccines 
The Journal of pediatrics  2009;155(4):495-499.
Objective
We conducted a population-based pharmacokinetic study to assess blood levels and elimination of mercury following vaccination of premature infants born at ≥ 32 and < 37 weeks of gestation and with birth weight ≥ 2000 but < 3000 grams.
Study design
Blood, stool, and urine samples were obtained pre-vaccination and 12 hours to 30 days post-vaccination from 72 premature newborns. Total mercury levels were measured by atomic absorption.
Results
The mean ± standard deviation (SD) birth weight was 2.4 ±0.3 Kg for the study population. Maximal mean ± SD blood mercury levels was 3.6 ± 2.1 ng/mL occurring at 1 day after vaccination, maximal mean ± SD stool mercury levels were 35.4 ± 38.0 ng/g, occurring on day 5 after vaccination, and urine mercury levels were mostly non-detectable. The blood mercury half-life was calculated to be 6.3 (95% CI:3.85-8.77) days and mercury levels returned to pre-vaccination levels by day 30.
Conclusions
The blood half-life of intramuscular ethyl mercury from thimerosal in vaccines given to premature infants is substantially shorter than that of oral methyl mercury in adults. Because of the differing pharmacokinetics, exposure guidelines based on oral methyl mercury in adults may not be accurate for children who receive thimerosal-containing vaccines.
doi:10.1016/j.jpeds.2009.04.011
PMCID: PMC2767297  PMID: 19560158

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